1. Field of the Invention
The invention relates to a saliva immunoassay for detection of antibodies for cardiovascular disease.
2. Description of the Related Art
Cardiovascular disease is predicted to be the most common cause of death worldwide by the year 2020. Half of heart disease patients lack established risk factors such as elevated lipids, hypertension, tobacco abuse, and positive family history. Additionally, these risk factors are generally associated with the disease, and the exact mechanism by which they may contribute to the development of atherosclerosis is not clear. However, previous and recent studies point to a linkage between infection with different bacteria and heart disease in the other 50% of observed incidences. Pathogenesis of the disease induced by infectious agents is described by three different mechanisms of action: release of toxins or superantigens, induction of inflammation, and molecular mimicry or cross-reactivity. This may result in plaque formation or antimyosin cellular and humoral immunity and subsequently, to myocarditis or other autoimmune diseases.
Through the years, many reports have incriminated various infectious agents in the pathogenesis of autoimmune disease. Moreover, the American College of Cardiology has issued a list of harmful pathogens as possible links to heart disease.
Traditionally, it is assumed that infectious agents induce disease by direct tissue damage via secretion of toxins or different antigens, particularly myosin. These toxins may directly or indirectly induce tissue damage and cause release of tissue antigens.
An infectious agent can be taken up by macrophages and transferred to the bloodstream and arteries. When a macrophage burrows into the wall of a blood vessel to take in irritants such as LDL and oxidized LDL, it transfers the infectious agent into the neighboring arterial cells. Infected arterial cells then attract more macrophages and other inflammatory responses, such as platelets, and then die. If this vicious cycle of inflammation continues, it can result in fibrous lesions or plaque formation. When pieces of the plaque break loose, they can start blood clots and cause heart attack.
Another mechanism by which infectious agents can cause autoimmune disease is molecular mimicry. Molecular mimicry is defined as structural similarity between antigens coded by different genes. Antigenic cross-reactivity between host and bacteria is exemplified by blood group substances and bacterial polysaccharides; cardiac tissue and streptococcal proteins; and kidney tissue and E. coli polysaccharides. Viruses may also induce autoimmune responses through shared determinants on molecules notably present on host cells, by altering the host immune system, or by causing the expression or release of “normally sequestered” self antigens.
Harmful pathogens may be the cause of many human diseases. These pathogens may induce their pathologic response through one of the above-mentioned mechanisms of action.
Many viruses, bacteria, and even parasites are claimed to affect atherosclerosis plaque deposition. Among them, Chlamydia pneumoniae probably has the strongest association with atherosclerosis. There is a close relationship between C. pneumoniae infection, IgG and IgM titers, and increased evidence of MI, CVA, and peripheral vascular disease (PVD). C. pneumoniae antigens are found in atherosclerosis plaques, and T-cell reactions to these antigens have been demonstrated. Experimental models illustrate the pathogenic role of C. pneumoniae and the unique heat shock protein (HSP)-60. Other major atherosclerosis-associated pathogens are Helicobacter pylori, Epstein-Barr virus and cytomegalovirus. For some pathogens, interfering pathogenic mechanisms have been described, such as cytomegalovirus gene-induced proliferation of smooth-muscle cells. From data showing a correlation between increased atherosclerosis incidence and chronic bronchitis, as well as periodontitis, it has been suggested that any infectious agent, and especially multiple chronic infections, could result in accelerated atherosclerosis formation. This multiplicity was confirmed recently in experimental animal models. There is no doubt therefore, that chronic infections with specific or nonspecific infectious agents can contribute to the acceleration of atherosclerosis development, either by nonspecific mechanisms [hypercoagulation and increased adhesion molecule and elevated C-reactive protein (CRP) levels] or by more specific mechanisms, such as induction of HSP-60 expression and eventually pathogenic anti-HSP-60 antibody production.
Autoantibodies are frequently found in the sera of virus-infected individuals, both during and after infection. For example, after infection with Epstein-Barr virus (EBV), antibodies reacting with intermediate filaments of cells, immunoglobulin or thyroglobulin were detected (Oldstone, J. Autoimmunity, 2(Suppl.):187-194, 1989; Srinivasappa et al., J. Virology, 57:397-401, 1986; Talal et al., J Clin. Invest., 85:1886-1871).
Myosin Antibody
Myosin accounts for over 50% of muscle proteins. Along with actin, myosin is involved in muscle contraction. Myosin is one of the largest proteins in the body, with a molecular mass of 500 kDa. Due to its large mass, antigenic mimicry between infectious agents and myosin molecules is highly probable.
It is now well-known fact that infectious agents are associated with human myocarditis. The development of autoimmunity to myocardial antigens has been widely recognized after myocardial infarction or after cardiac surgery. Autoantibodies to heart tissue in patients with rheumatic carditis, post-myocardial infarction and post-pericardiotomy syndromes have been described. Antibodies against heart tissue can also occur in patients with post-infection myocariditis, dilated cardiomyopathy, rheumatic carditis, Chagas disease, and adriamycin cardiotoxicity. It has also been observed that serum from patients with myocarditis reacted specifically with sarcolemmal and cytoplasmic heart antigens. Moreover, serum samples containing circulating heart antibodies also induced complement-mediated myocyte lysis and antibody-dependent cell-mediated cytotoxic reactions in vitro, suggesting that they may be pathogenic in myocarditis.
It is now clear that some patients with active myocarditis or cardiomyopathy carry antibodies to the mitochondrial adenine nucleotide translocator. Such patients, whose serum inhibit in vitro ADP-ATP translocator activity, have reduced cardiac function relative to their counterparts without these antibodies. The existence of multiple heart-reactive antibodies in autoimmune heart disease is consistent with the presence of multiple tissue- and organelle-specific antibodies in both systemic lupus and autoimmune thyroiditis.
For years it has been known that Chlamydia can induce cardiovascular disease in experimental animals. This Chlamydia-mediated heart disease in mice can be induced by antigenic mimicry of a heart muscle-specific protein, thus providing a molecular link between Chlamydia infections and heart disease. Since many infectious agents have been implicated in heart disease, it is not surprising that organisms other than Chlamydia can also supply mimicking epitopes. Indeed, Machmaier, K. et al., in a study published in Nature Medicine in August 2000, screened public databases for proteins sharing the pathogenic mouse M7Aα peptide MA'ST motif (whose amino acid sequence is as follows: SLKLMATLFSTYASA). This motif is found in proteins from a multitude of viruses, bacteria, fungi, and protozoa, which are involved in cardiovascular disease.
Cross-reactive antibodies appear to be quite common in patients with rheumatic fever. Some of these autoantibodies could be absorbed by certain streptococcal strains, and some reacted specifically with cardiolipin and tropomyosin. Group A and mutant streptococci share a common epitope with cardiac myosin, which may be associated with the heavy meromyosin region of the molecule. In Chagas disease—caused by the protozoan parasite Trypanosoma cruzi—heart autoantibodies react with laminin, while Chagasic cardiomyophathy may be due to recognition of the calcium-sequestering ATPase in the sarcoplasmic reticulum.
Oxidized LDL Antibody
Oxidized Low Density Lipoprotein (oLDL), the prime candidate for an autoantigen, plays a critical role in the development and progression of atherosclerosis and other vascular diseases. It is incriminated in foam cell generation through uptake by the unregulated scavenger receptors on macrophages.
Recent evidence suggest that autoantibodies against oxidatively modified LDL can be used as a parameter that consistently mirrors the occurrence of oxidation processes taking place in vivo. In fact, elevated levels of autoantibodies against oLDL have been detected in the bloodstream of patients with coronary artery disease. Moreover, recent studies indicate a correlation between autoantibodies against oLDL and the progression of carotid atherosclerosis. Increased serum concentrations of oLDL have also been described in various diseases such as pre-eclampsia and systemic lupus erythematosus (SLE).
Heat Shock Protein 60 (HSP60) Antibody
Heat Shock Protein 60 (HSP60), also known as CPN60, is an abundant protein synthesized constitutively in the cell that is induced to a higher concentration after brief cell stress or shock. It is present in all species analyzed so far and exhibits a remarkable sequence homology among various counterparts in bacteria, plants, and mammals: more than half of the residues are identical between bacterial and mammalian HSP60. The ubiquitous occurrence and remarkable evolutionary conservation suggests that HSP60 may play an essential role in the cell. It is now believed that HSP60, which is localized in mitochondrial matrix in eukaryotes, interacts with multiple proteins during translocation and/or folding. E. coli HSP60 (GroEL) has been shown to catalyze folding of many proteins in vitro and is involved in the assembly of bacteriophage lambda proteins during infections. TCP-I, a member of the HSP60 family, has similar functions to HSP60 but is localized within the cytoplasm. Bacterial HSP60 proteins are major targets of immune responses during infection, and the highly conserved nature of bacterial and mammalian HSP60 has led to speculation that immune reactivity to these stress proteins may be a component of certain autoimmune diseases and atherosclerosis. In fact, G. Wick (Innsbruck, Austria) first claimed that HSP60 is involved in atherosclerosis. Anti-HSP60 antibody titers correlate with the degree of atherosclerosis in carotid ultrasound studies. The increase in anti-HSP60 antibody levels could result from direct turbulence damage to bifurcated arteries or could be caused by infectious agents (e.g. C. pneumoniae) releasing HSP60, which becomes immunogenic. T-cell lines cultured from the atherosclerosis plaque proliferate when exposed to HSP60 and both the autoantibodies, as well as the autoantigen can be found in the plaque. Finally, active immunization of rabbits and apolipoprotein-E or low-density lipoprotein (LDL)-receptor knockout mice with HSP60 leads to accelerated formation of atherosclerosis plaques.
Anti-β2-Glycoprotein-1
β2-Glycoprotein-1 (p2GP1) is a normal glycoprotein synthesized by the liver that behaves as an anti-coagulant and is also an anti-atherogenic agent. This glycoprotein, also known as apolipoprotein-H, is a human plasma glycoprotein that consists of a single polypeptide of 326 amino acids with a molecular weight of 50 kDa.
It is now widely accepted that β2GP1 is an absolute requirement for the binding of “antiphospholipid” (aPL) Abs purified from patients with autoimmune disease when assayed using anionic phospholipid ELISAs. These autoantibodies are of considerable clinical importance because of their association with arterial and venous thrombosis, recurrent fetal loss, and thromobocytopenia. The interaction of autoantibodies with β2GP1 may be important in relation to the pathogenesis of thrombosis in vivo. β2GP1 is known to bind to negatively charged surfaces as well as to activated platelets and to act as an inhibitor of the intrinsic blood coagulation pathway in vitro.
β2GP1 also binds to oLDL. This binding of β2GP1 to oLDL reduces the uptake of oLDL by scavenger receptors on macrophages. In fact, β2GP1 is found in the atherosclerosis plaque and is the target antigen in antiphospholipid syndrome (APS). Antibody titer to β2GP1 correlates with atherosclerosis. In in vitro conditions, these antibodies enhance uptake of oLDL by macrophages.
Recently, in a classical study, accelerated atherosclerosis plaque formation was induced in LDL-receptor-deficient mice by the passive transfer of lymphocytes from the lymph nodes and spleens of mice actively immunized with β2GP1.
Anti-Platelet Glycoproteins
A number of diseases and syndromes are thought to involve antibody, or immune complex-mediated platelet destruction. Among these are both the acute and chronic forms of idiopathic thrombocytopenic purpurea; the closely related thrombocytopenia of systemic lupus erythematosus; quinidine, apronalide, and other drug-induced thrombocytopenias; post-transfusion purpurea; neonatal isoimmune thromobocytopenia; and the alloimmunization that renders multi-transfused patients refractory to random platelet transfusion.
Platelet function and number can both be affected in immune-mediated diseases; however, thrombocytopenia is by far the more common finding. Abnormalities of platelet number and function can occur via any of several immune mechanisms. Both humoral and cell-mediated immune mechanisms can produce thrombocytopenia. The most commonly considered, although by no means the most commonly noted, immune mechanism for thrombocytopenia is the formation of specific antiplatelet autoantibodies. Platelets have a large number of immunogenic structures on their surface, with the glycoprotein IIb/IIIa (GP IIb/IIIa) complex being the most numerous. It is not surprising, therefore, that autoantibodies directed against epitopes on the GP IIb/IIIa complex are the most frequent when the specificity of the autoantibodies have been determined in blood. Platelet autoantibodies are usually of the IgG immunoglobulin class, although IgA, IgD, and IgM autoantibodies have been demonstrated occasionally. Complement has also been found on surface of platelets in clinical syndromes consistent with increased immune-mediated platelet destruction. However, most autoantibodies are not complement fixing, and removal of the immunoglobulin-coated platelets occurs in the spleen and other sites of reticuloendothelial tissue.
Immune Complexes
Immune complexes are formed when antigens bind with antibodies. Antigen-antibody complexes can activate the complement cascade and bind the C1q component of complement and form pathologic complexes.
Both exogenous and endogenous antigens can trigger pathogenic immune responses that result in immune complex (IC) disease. Because circulating IC's play such an important part in many diseases, including autoimmunity, neoplasms, infectious diseases due to bacteria, viruses, and parasites, and other unclassified disorders, the demonstration of IC's in tissues and biological fluids has achieved rising prominence.
There are a number of cases in which immune complexes assays are helpful in the diagnosis and monitoring of disease activity, for example, lupus and arthritis.
The fact that SLE is considered the prototype of human immune complex disease has led to studies of SLE with almost every type of immune complex assay developed. A high incidence of positive tests and disease activity has been uniformly reported. There is considerable evidence that DNA-anti-DNA complexes are involved in the pathogenesis of SLE. Immune complex determinations coupled with detection of serum antibodies to native DNA and determinations of levels of hemolytic complement (CH50) in serum are useful diagnostic tests. Most studies have found a correlation between positive immune complex assays and antibodies to native DNA, which is the most important laboratory marker of lupus. Several serial studies have indicated that the C1q solid-phase assay correlates better with disease activity than do other immune complex tests.
The role of circulating immune complexes (CIC) in cancer is of particular interest because tumors express antigens that elicit both cellular and humoral immune responses. CMI in tumor-bearing host is blocked by CIC or “blocking factors” in circulation. Antigen-antibody complexes are formed by noncovalent hydrophobic coulombic hydrogen bonds. The nature and quantity of CIC detected in circulation is dependent upon the dynamics of formation, clearance, and tissue deposition of immune complexes. Immune complexes cause tissue injury through the terminal lytic component of activated complement system. Since activated complement components are strong chemotactic agents, leukocytoclastic vasculitis is seen in cancer patients with high levels of CIC.
Manifestation of Antibodies
The deposition of antigens in the gut has been shown to lead to the production of IgA antibodies in secretions at sites distant from the gut, such as colostrums, lacrimal and salivary secretions in man and salivary secretions in rhesus monkeys and in rats.
A general conclusion therefore is that the secretory immune system can be stimulated centrally and that precursors of IgA-producing cells migrate from the gut-associated lymphoid tissue to several secretory sites in addition to the lamina propria of the gut itself. Therefore, if antigens are injected into the submucosal tissues, they are likely to induce serum IgG antibodies as well as secretory IgA antibodies in saliva. However, if it is applied topically to the skin or to the intraepiethelial tissue, secretory IgA is the main product which is detected in saliva. The role of topically applied antigen in the localization and persistence of IgA responses has been demonstrated in several secretory sites, including the respiratory tract, oral cavity, gut, and vagina.
The evidence that cells migrate from the gut to various secretory tissues, and that immunization in the gut leads to antibodies at various secretory sites has led to the concept of a common mucosal system. However, this concept may be an oversimplification, since although immunization in the lung may lead to antibodies in distant secretory sites, such as salivary glands and immunization in the lacrimal glands has also been shown to lead to the production of antibodies in saliva. Thus, with firm evidence that antigen deposition in the gut may lead to antibodies not only in the gut but also in saliva, lungs, lacrimal secretions and genitourinary tract, it is probably more correct to designate the system as an enteromucosal system.
Saliva is a source of body fluid for detection of an immune response to bacterial, food, and other antigens present in the oral cavity and gastrointestinal tract. Indeed, salivary antibody induction has been widely used as a model system to study secretory responses to ingested material, primarily because saliva is an easy secretion to collect and analyze. It seems to be a general feature that salivary IgA antibodies can be induced in a variety of species in the absence of serum antibodies. This has been demonstrated after immunization with particulate bacterial antigens in human could selectively induce an immune response to Streptococcus mutans by oral administration of the antigen. This route of administration resulted only in antibody production in saliva and not in serum. Similar mucosal immune response in the form of saliva IgA did occur in monkeys, rabbits, rats, and mice after oral administration of Streptococcus mutans or other bacteria.
This lack of production of IgG, but IgA production in saliva after oral or intragastric administration of bacterial antigens is shown in the following table.
TABLE 1Induction of salivary IgA antibody after stimulationof gut associated lymphoid tissueSalivarySerumRoute ofIgAAntibodySpeciesAntigenAdministrationProductionProductionHumanStreptococcusOral++−MutansMonkeysStreptococcusIntragastric++−MutansRabbitsPenumococcusIntragastric++−or BGGRatsStreptococcusOral++−MutansMiceStreptococcusIntragastric++−Mutans orOvalbumin
As indicated in this table, oral or intragastric administration of dietary soluble proteins such as bovine gammaglobulin (BGG) and ovalbumin or eggalbumin resulted in salivary IgA production but not in any antibody production in serum. For these reasons, saliva has been selected not only because of its relevance in oral disease, but mainly because it is an accessible fluid, easy to collect, and is thought to show representative responses in secretions after central or intragastric immunization. However, if both saliva IgA and serum IgG antibodies are detected in the same patient, it means that this individual has been primed with the antigen orally as well as systematically.
This IgA production in saliva and IgG production in serum is dependent upon antigen dosage as well as the integrity of the gut. For example, a single intragastric immunization with 1 mg of eggalbumin led to oral tolerance but did not lead to detectable secretory IgA antibodies, whereas 10 mg of ovalbumin led to systemic tolerance, but to a significant level of salivary IgA antibodies. Thus, detection of high levels of antibody in saliva is an indication of the body's exposure to significant levels of antigenic stimulation.
While this concept of oral tolerance to high doses of soluble antigen may be correct, certain conditions—such as overloading of the GI tract with bacterial toxins—may not lead to oral tolerance. This is due to the fact that bacterial toxins will cause the opening of tight junctions, which will in turn lead to the absorption of ingested proteins and bacterial antigens from the gut in significant amounts. This excessive uptake of bacterial, fungal, viral, and dietary proteins into the circulation may induce immune response first in the form of IgM, and thereafter in the form of IgG and IgA antibodies in the serum, all of which may lead to different clinical conditions.